Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring cutaneous lesions (e.g., L. mexicana) or degenerative and most often fatal visceral disease (e.g., L. donovani ). These diseases afflict over 12 million patients in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of this organism. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membranes seems essential. To that end, unique parasite surface membrane enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized, for the first time, toward defining their expression and regulation during course of parasite growth, differentiation and development. During the past year, our studies have elucidated the gene structure and chromosomal loci of several different members of the unique trypanosomatid surface membrane 3?-nucleotidase/ nuclease family from L .donovani, L. mexicana and Crithidia luciliae. Various mutated-expression constructs of these genes were made and used to identify the specific regions of these proteins responsible for their enzymatic activities and for their proper targeting to the parasite cell surface. Several of these mutated expression-constructs have also proven useful for the laboratory-scale production and purification of these normally membrane-bound parasite enzymes in a soluble form. Similar studies are in progress with the unique leishmanial chaperone protein, Ld-calreticulin and surface membrane acid phosphatase. Further, experiments involving the targeted deletion of these genes and the production of functionally null mutant parasites are in progress. The viability of such mutant parasites will be tested in situ. Results of such studies should demonstrate whether the proteins encoded by these genes are, in fact, essential for the survival of these pathogens within their insect-vector and/or mammalian hosts. Thus, the current studies are aimed at defining the intrinsic needs of these organisms and determining their critical nature. Results of our recent and ongoing studies continue to provide pertinent information toward understanding the unique pathophysiology of these organisms. In addition, these studies are of practical relevance toward demonstrating whether such parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs and 2) the development of new diagnostic tools. They are also relevant for determining whether they are useful as potential vaccines against these human pathogens.